Cutaneous leishmaniasis (CL), a disease caused by parasites of the Leishmania genus and transmitted by sand flies, is considered by the WHO to be a neglected tropical disease, affecting about 1.5 million people per year. Approximately 70% of CL cases are reported from six countries, including Iran, where CL is endemic in 17 out of 30 provinces, with over 20,000 cases reported annually; the actual incidence has been estimated to be five times higher. There is no vaccine to prevent this disease. Efforts to control zoonotic CL (ZCL), including application of indoor residual insecticides, poisoning of rodent reservoirs, and use of insecticide-treated curtains and bednets (ITNs) have had limited success. In this 2-year project, we propose a novel field-based strategy aimed at prevention of Leishmania major transmission by the sand fly, P. papatasi, with a focus on the hyper-endemic region of Isfahan Province, Iran. Using the sand fly gut-associated bacterium, E. cloacae dissolvens, which has been isolated from biotic loci of ZCL in Isfahan, we propose to develop a paratransgenic model that may eventually be applied to field control. Initially, E. cloacae will be transformed to express the anti-leishmanial peptides, melittin and histone H2B, which act synergistically in vitro to kill L. major at concentrations as low as 1-3 M. Engineered E. cloacae will then be delivered to laboratory lines of P. papatasi using a soil-based strategy that targets larval stages; production of the recombinant peptides will be verified in adult flies. Challenge of the adult female flies with L. major is expected to result in parasite-refractory insects. Additional studies, to be conducted at Tehran University of Medical Sciences (TUMS), will simulate microbial conditions of rodent burrows where P. papatasi breed to establish the infectivity and persistence of the engineered bacteria in flies under conditions that mimic field settings. Final project deliverables will include engineered lines of E. cloacae dissolvens and a laboratory model of paratransgenic P. papatasi that can be further developed for field deployment in Iran and neighboring regions of the Middle East.